Advanced Higher Biology

Risk

the likelihood of harm arising from exposure to a hazard

Risk assessment

Identifying control measures to minimise the risk

Hazards

Toxic or corrosive chemicals, heat or flammable substances, pathogenic organisms, and mechanical equipment.

Control measures

Using appropriate handling techniques, protective clothing and equipment, and aseptic technique.

Linear dilution series

Dilutions differ by an equal interval, for example 0·1, 0·2, 0·3 and so on

Log dilution series

Dilutions differ by a constant proportion, for example 10-1 , 10-2 , 10-3 and so on.

What does plotting measured values for known concentrations to produce a line or curve allow?

The concentration of an unknown to be determined from the standard curve

How do buffers control pH?

Addition of acid or alkali has very small effects on the pH of a buffer, allowing the pH of a reaction mixture to be kept constant

How to use a colorimeter to quantify concentration and turbidity

Calibration with appropriate blank as a baseline

Use of absorbance to determine concentration of a coloured solution using suitable wavelength filters

Use of percentage transmission to determine turbidity, such as cells in suspension.

How does a centrifuge separate substances of differing density?

More dense components settle in the pellet

Less dense components remain in the supernatant

Paper and thin layer chromatography can be used for separating different substances such as…

Amino acids and sugars

What does the speed that each solute travels along the chromatogram depend on?

It’s differing solubility in the solvent used.

How is affinity chromatography used to separate proteins?

A solid matrix or gel column is created with specific molecules bound to the matrix or gel. Soluble, target proteins in a mixture, with a high affinity for these molecules, become attached to them as the mixture passes down the column. Other non-target molecules with a weaker affinity are washed out.

What is gel electrophoresis used to do and how does it work?

Separating proteins and nucleic acids

Charged macromolecules move through an electric field applied to a gel matrix

What are native gels and how do they work?

Native gels separate proteins by their shape, size and charge

Native gels do not denature the molecule so that separation is by shape, size and charge

What is SDS-PAGE and how does it work?

SDS–PAGE separates proteins by size alone

SDS–PAGE gives all the molecules an equally negative charge and denatures them, separating proteins by size alone

What is an isoelectric point (IEP) ?

IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.

Proteins can be separated from a mixture using their isoelectric points (IEPs). How?

IEP is the pH at which a soluble protein has no net charge and will precipitate out of solution.

If the solution is buffered to a specific pH, only the protein(s) that have an IEP of that pH will precipitate

Proteins can also be separated using their IEPs in electrophoresis. How?

Soluble proteins can be separated using an electric field and a pH gradient. A protein stops migrating through the gel at its IEP in the pH gradient because it has no net charge

What are immunoassay techniques are used for and what do they use?

To detect and identify specific proteins

These techniques use stocks of antibodies with the same specificity, known as monoclonal antibodies

An antibody specific to the protein antigen is linked to what?

A chemical ‘label

The ‘label’ is often a reporter enzyme producing a colour change, but chemiluminescence, fluorescence and other reporters can be used

In some cases the assay uses a specific antigen to detect the presence of antibodies

What is western blotting?

Western blotting is a technique, used after SDS–PAGE electrophoresis The separated proteins from the gel are transferred (blotted) onto a solid medium

The proteins can be identified using specific antibodies that have reporter enzymes attached

Bright-field microscopy is commonly used to observe what?

Whole organisms

Parts of organisms

Thin sections of dissected tissue

Individual cells

What is aseptic technique?

Aseptic technique eliminates unwanted microbial contaminants when culturing microorganisms or cells

Aseptic technique involves the sterilisation of equipment and culture media by heat or chemical means and subsequent exclusion of microbial contaminants

How can a microbial culture be started?

A microbial culture can be started using an inoculum of microbial cells on an agar medium, or in a broth with suitable nutrients

Many culture media exist that promote the growth of specific types of cells and microbes

Animal cells are grown in medium containing growth factors from serum. What are growth factors?

Growth factors are proteins that promote cell growth and proliferation

Growth factors are essential for the culture of most animal cells

In culture how frequently can both primary cell lines and tumour cell lines divide?

In culture, primary cell lines can divide a limited number of times, whereas tumour cells lines can perform unlimited divisions

What does plating out of a liquid microbial culture on solid media allow?

Plating out of a liquid microbial culture on solid media allows the number of colony forming units to be counted and the density of cells in the culture estimated

Serial dilution is often needed to achieve a suitable colony count

What are haemocytometers are used for?

Haemocytometers are used to estimate cell numbers in a liquid culture

What is vital staining required for?

To identify and count viable cells